One of the most fundamental principles in biology is the relationship between DNA, RNA and proteins. Generally, in eukaryotic organisms, DNA comprises genes, which can be transcribed into messenger RNA (mRNA) molecules, and these mRNA molecules are subsequently translated into proteins. These different steps take place at different locations in a eukaryotic cell: the DNA is contained inside an organelle which is located inside the cytoplasm of a eukaryotic cell (i.e. a nucleus, mitochondrion or chloroplast). However, the mRNA transcript needs to be exported out of that organelle, because translation typically takes place in the cytoplasm of the cell, which is where the ribosomes required for translation are located.
Thus, it is fundamental for the normal functioning of biological systems that mRNA transcripts are transported across organellar membranes, from one subcellular compartment to another subcellular compartment. In this manner, a cell's genetic information is efficiently translated into a large number of different proteins that work together to enable the cell to maintain its own functionality and metabolism, and to respond adequately to its environment and to signals from other cells in the organism, and to signals from the organism's environment. This so-called cell-autonomy thus leads to a situation in which a cell's phenotype and behaviour is directly linked to that cell's own genetic information, in a multicellular organism.
However, researchers have also observed cases of non-cell-autonomy, wherein one cell influences the phenotype of another cell. In a multicellular organism, different cells communicate with each other, and thus they influence and coordinate each other's behaviour and phenotype. In some cases, a mutation present in the genome of one cell may even influence the phenotype of another cell that does not have said mutation in its genome.
A good example of non-cell-autonomy has been observed in plants. In the phloem sap of plants, many protein-coding mRNA transcripts, small RNA molecules and proteins have been detected that can move from one location in the plant to another location. Thus, in some instances one cell produces an mRNA transcript, and then exports it to another cell, where the mRNA transcript may be translated into a protein, and where the protein fulfils its function. This exchange of information over long distances, from one cell-type, tissue or organ to another via the vascular tissue, is very poorly understood, but it provides a new level of complexity in how plants function, how they coordinate their own development and growth, and how their organs exhibit a concerted response to the plant's environment. It reveals that cells and organs in different parts of the organism communicate with each other, not only by means of small signalling molecules such as hormones, but also by exchanging macromolecules in a coordinated manner. The mode of action of this system (RNA motifs that trigger mobility, the extent of their transport, and their potential to be translated into functional proteins after transport) is however unknown in the prior art.
In the research leading to the present invention, it was observed that many mRNA transcripts that are present in the phloem sap of plants (and that are enriched in the pool of mRNA transcripts that move across chimeric graft junctions) comprise a tRNA-like structure (TLS). It was demonstrated that tRNA-like structures are sufficient to mobilise mRNA transcripts, by showing that mRNAs harbouring distinctive TLS move from transgenic roots into wild-type leaves and from transgenic leaves into wild-type flowers or roots, when wild-type and transgenic plants are grafted onto each other. It was furthermore shown that these mobile mRNA transcripts are translated into proteins after their transport. It was also found that bicistronic mRNA::tRNA transcripts (i.e. mRNA transcripts harbouring a TLS) are frequently produced in Arabidopsis thaliana, and that they are enriched in the population of graft-mobile mRNAs. “Graft-mobile mRNAs” are mRNAs that are capable of moving across a graft junction.
RNA molecules are arguably the most functionally diverse biological macromolecules found in cells, and their diverse roles are determined by both their complex three-dimensional structure and by their primary sequence. An additional biological role of tRNA sequences (or tRNA-like structures, TLSs) has now been uncovered in plants: they harbour a motif mediating mRNA transport to distant plant cells. Interestingly, transcript mobility was induced by tRNAMet and tRNAGly, but not by one particular tRNAIle, which correlates with the absence of this tRNAIle in pumpkin phloem sap. As the present results indicate that transcript mobility is mediated by a particular RNA structure, a tRNA motif-scanning algorithm did indeed reveal that a significantly high number of identified mobile mRNAs harbours a TLS motif or is transcribed by genes in close proximity of annotated tRNA genes which seem to frequently produce bicistronic poly(A)-RNA::tRNAs.
While the functional role of many mobile mRNAs in distant tissues remains to be elucidated, evidence supports the notion that trafficking of small si/miRNA and large mRNAs via the phloem plays an important role in regulating plant development. A surprisingly high number of mRNAs is present in phloem exudates and was shown to move across graft junctions, but no general and easily predictable RNA motif or conserved sequence mediating mobility could be identified in the graft-mobile transcript populations. However, the present invention now demonstrates that a significant fraction of mobile mRNAs carries a TLS motif that mediates mobility across graft junctions.
Messenger RNA transfer does not strictly follow the source to sink phloem flow, as it could be demonstrated that GUS::tRNA fusions not only move from shoot (source) to root (sink), but also vice versa. Two transport pathways were found to exist for delivering mRNA molecules. One is based on passive, non-selective delivery from source to sink via the phloem vessels. However, according to the invention also another pathway in form of a targeted and active transport system was found. This pathway inter alia mediates the delivery of mRNAs from root to shoot. Presence of an active mRNA delivery mechanism is supported by two findings according to the invention, namely that sequences derived from TLS are sufficient to confer mobility to heterologous mRNAs, and that deletion of a TLS in the plant endogenous CK1::tRNAGly bicistronic transcript, which is naturally mobile, makes it immobile.
It was shown according to the invention that TLSs or closely related RNA structures mediate transport of a number of graft-mobile transcripts, and that specific tRNA sequences such as tRNAGly-, tRNAMet-, and tRNAMet-derived sequences trigger transport of otherwise non-mobile transcripts, and that a significant number of mobile mRNAs harbour a TLS motif.
The invention is thus based on the fact that this structural motif (TLS, with predicted stem-bulge-loop structures that are identical or very similar to a tRNA) plays a role in the coordinated long-distance transport of mRNA transcripts.
In animals, evidence for long-distance intercellular mRNA transport also exists. In human plasma, for example, the presence of cell-free mRNA of the hTERT and MUC1 genes has been found to correlate with gastric cancer (Tani et al., 2007, Anticancer Res. 27: 1207-1212). Both genes have been linked to oncogenesis in humans. It is conceivable that the occurrence of cell-free mRNA of these genes in plasma represents a mode of communication between cancer cells and healthy cells. If healthy cells have a means of uptake for these mRNA transcripts, they may start producing the encoded proteins. Mammalian cells have been reported to be capable of taking up exogenous mRNA (see, for example, Probst et al., 2007, Gene Ther. 14: 1175-1180). Various mutations in hTERT have been linked to an increased risk of various cancers, and by sending out mRNA transcripts comprising such mutation, one single cell carrying an oncogenic mutation in its genome (e.g. as the result of a non-inherited de novo mutation) may have the capacity to distribute its mutated version of the mRNA transcript to many other cells that may not harbour said mutation in their own genome, but that are receptive for taking up said transcript from the extracellular matrix. In such a manner, healthy cells will produce a mutant (and potentially oncogenic) form of the encoded protein, even when their own, endogenous gene coding for that protein is not mutated. In the case of MUC1, it has been shown that up-regulation of this gene is associated with cancer (Zaretsky et al., 2006, Mol. Cancer 5: 57). Here again, the presence of mRNA transcripts of this gene in blood plasma would provide an opportunity for cells to take up these cell-free mRNA transcripts and to produce the proteins encoded thereby, independently of the promoter activity of their own, endogenous MUC1 gene. This may lead to a higher than normal production of this protein in many cells in the plasma, which, in turn, may be linked to oncogenesis.
The occurrence of disease-specific mRNA transcripts in blood has also been observed in, for example, patients with the neurodegenerative disorder Alzheimer's disease (Koh et al., 2014, Proc. Nat. Acad. Sci. USA 111: 7361-7366). This mRNA secretion from neurons may represent a way in which this disorder progresses and spreads between the neurons of a patient.
In animal cells, proteins and RNA may be secreted from cells through vesicles in an unconventional secretion mechanism, which is different from the Endoplasmic Reticulum (ER)/Golgi pathway. It is possible that TLSs play a role in the selective loading of mRNA transcripts into vesicles and/or in the specific secretion of mRNA from cells into the extracellular matrix (e.g. blood, plasma). If the presence of a TLS also enables an mRNA transcript to be taken up by other cells, said TLS would essentially mediate cell-to-cell movement of mRNA transcripts in animals, similarly to what has been observed in plants in the research leading to the present invention.
The invention thus relates to a method for changing the intercellular mobility of an mRNA of a gene in an organism, comprising:                modifying a tRNA-like structure (TLS) present in the mRNA by mutating the gene from which the mRNA is transcribed, or        including the sequence of a tRNA-like structure (TLS) in the transcribed part of the gene.        
There are essentially three ways in which the present invention can be applied to change the intercellular mobility of an mRNA of a gene in an organism. On the one hand, the mobility of an mRNA transcript that is mobile in a wild-type organism can be prevented, by modifying the tRNA-like structure that is naturally present in said mRNA transcript in such a way that it no longer promotes mobility of the mRNA transcript.
On the other hand, an mRNA transcript that is not mobile in a wild-type organism can be made mobile, by providing said mRNA transcript with one or more tRNA-like structures that have the ability to promote mobility of the mRNA transcript. This embodiment can be applied to mRNA transcripts from genes that are endogenous to the organism, but also to mRNA transcripts from genes that are not endogenous to the organism, such as genes that have been introduced into the organism's genome by means of genetic modification, or that have been introduced into the organism in a transient manner, e.g. by injection, ingestion, transfection, or by exogenous application of mRNA molecules comprising a TLS on the organism's surface, and that are subsequently transported further inside the organism and imported into cells by virtue of the presence of said TLS.
In a third embodiment, the mobility of an mRNA transcript can be changed in the sense that the destination of the transcript inside the organism (i.e. the cell-type, tissue or organ where the mRNA transcript moves towards in a wild-type organism) changes as a result of modifications in the TLS. In case different destinations in the organism are linked to different TLSs, modifying the original TLS of an mRNA can direct the transcript to a different destination inside the organism, which results in an ectopic production of the encoded protein.
Modification of TLSs and inclusion of such TLSs in transcripts can be achieved by means of genetic modification. Genetic modification comprises transgenic modification or transgenesis, using a gene from a non-crossable species or a synthetic gene, and cisgenic modification or cisgenesis, using a natural gene, coding for a trait from the organism itself or from a sexually compatible donor organism.
In one embodiment, the organism of which the intercellular mobility of the mRNA of a gene is to be changed is a multicellular eukaryotic organism, such as a plant, a fungus, or an animal. In a further embodiment, the organism is a dicotyledonous plant, a monocotyledonous plant, a gymnosperm, a bryophyte, an algae, a mammal, a non-mammalian vertebrate animal, or an invertebrate animal.
In the context of this invention, the term “organism” is intended to not only comprise living multicellular plants, animals and fungi, but also tissue cultures and cell cultures that have been derived from living multicellular plants, animals and fungi. Just like a living organism, said tissue cultures or cell cultures consist of multiple cells, between which cells mRNA can be exchanged.
According to a first aspect of the invention, said organism is a plant. In one embodiment, the organism is a plant belonging to one of the following plant genera, or a cell culture or tissue culture derived therefrom: Allium, Apium, Beta, Brassica, Capsicum, Cichorium, Citrullus, Cucumis, Cucurbita, Benincasa, Daucus, Eruca, Lactuca, Lagenaria, Luffa, Phaseolus, Pisum, Lens, Raphanus, Solanum, Spinacia, Valerianella, Nicotiana, Petunia, Arabidopsis, Capsella, Arabis, Cardamine, Malus, Pyrus, Prunus, Vitis, Rosa, Fragaria, Populus, Fagus, Pinus, Picea, Ginkgo, Larix, Betula, Quercus, Salix, Alnus, Corylus, Amygdalus, Vaccinium, Rubus, Persea, Citrus, Castanea, Acer, Fraxinus, Coffea, Camellia, Theobroma, Olea, Cicer, Juglans, Pistacia, Arachis, Anacardium, Macadamia, Ficus, Litchi, Actinidia, Bougainvillea, Helianthus, Hibiscus, Malva, Glycine, Gossypium, Cannabis, Stevia, Opuntia, Ipomoea, Manihot, Humulus, Acacia, Medicago, Trifolium, Lotus, Vicia, Linum, Fagopyrum, Zea, Triticum, Avena, Hordeum, Oryza, Zizania, Secale, Triticosecale, Sorghum, Bambusa, Dendrocalamus, Saccharum, Cymbopogon, Pennisetum, Panicum, Festuca, Lolium, Phleum, Poa, Miscanthus, Asparagus, Agave, Yucca, Cocos, Elaeis, Phoenix, Amaryllis, Narcissus, Aloe, Canna, Iris, Colchicum, Crocus, Gladiolus, Juncus, Lilium, Tulipa, Musa, Dendrobium, Phalaenopsis, Vanilla, Typha, Zingiber, Curcuma, Lemna. Cell cultures and tissue cultures derived from plants include, for example, microspore cultures, pollen cultures, callus cultures, root cultures, meristem cultures, protoplast cultures, mesophyll cell cultures, tissue or organ explants, and the BY-2 cell culture.
According to a second aspect of the invention, said organism is an animal. In one embodiment, said organism is a mammal, a non-mammalian vertebrate animal, or an invertebrate animal. In a further embodiment, the organism is a mammal belonging to one of the following genera, or a cell culture or a tissue culture derived therefrom: Mus, Homo, Rattus, Pan, Cricetus, Mesocricetus, Cavia, Canis, Oryctolagus. Mammalian cell cultures often used in research are, for example, CHO, HeLa, HEK293, HL-60, J558L, JY, K562, KBM-7, RBL, and COS-1.
In one embodiment, the intercellular mobility is between cells in the same organ of an organism, or between cells in the same tissue culture or cell culture. In this context, the concept of an “organ” also comprises, for example, the blood and lymph of animals. In another embodiment, the intercellular mobility is between cells in different organs of an organism.
Intercellular mobility of an mRNA of a gene in an organism may occur across the plasma membranes of neighbouring cells in a tissue, or it may occur across the plasma membranes of cells that are not positioned adjacent to each other in a tissue. In the latter embodiment, said mobility may take advantage of the organism's own means for long-distance transport and cell-to-cell communication and/or of the extracellular matrix, such as the vascular tissue (phloem and/or xylem) or the apoplast in plants, or the blood stream or lymphatic system in animals. Cells that are not positioned adjacent to each other in a tissue may for example be cells that belong to different tissues or organs in the organism, or cells that are not attached or connected to other cells, such as blood cells in an animal, pollen grains in a plant's anther, etcetera.
In the course of normal development and growth, the exchange of mRNA transcripts between cells appears to be commonplace. It may however also be a phenomenon that plays an important role in the establishment and/or progression of disease or in the organism's defence against disease, as it allows the transport of genetic material—in the form of an already transcribed mRNA—from one cell to other cells, even across large distances within an organism. This implies that the presence in an organism of a single cell that harbours in a gene in its genome a mutation that causes a certain effect in the protein encoded by said gene, may result in the export of the mRNA comprising said mutation to many other cells in the organism that—in their own genome—lack said mutation. This may thus lead to a situation in which cells lacking a mutation in their own genetic material import mRNA molecules comprising said mutation, and subsequently translate that mRNA molecule into the protein that is encoded by said mRNA molecule. The effect thereof is that cells lacking said mutation may produce and harbour a protein that comprises said mutation. The mRNA transcripts of one mutated cell could thus affect the behaviour and/or phenotype of a multitude of non-mutated cells in the same organism, if the mRNA molecules sent out by said mutated cell are mobile and are able to move from the cell by which they have been produced to another cell in the same organism where they can be translated into protein. This mechanism may e.g. be relevant in the blood stream of animals, where essentially all cells are in a position to communicate with each other by means of molecules that are present in the blood, and in plants, wherein all organs are connected to each other by means of the vascular tissue.
Alternatively, the one cell may not be mutated, but it may be subjected to a stimulus, which causes it to produce a specific mRNA transcript or a specific splice-form of an mRNA transcript. This specific transcript or splice-form thereof may subsequently be transported to other cells in the organism, which had not (yet) received that stimulus, or which are incapable of responding to the stimulus. In plants, for example, cells of a leaf may respond to a change in light quality, and signal this perception to the roots. Root cells may detect changes in water availability in the soil, and communicate this to e.g. stomatal cells. Mobile mRNA transcripts may constitute a part of such intercellular communication signals between different parts of an organism.
In one embodiment, the invention relates to a method for changing the intercellular mobility of an mRNA of a gene in an organism, comprising modifying a tRNA-like structure present in the mRNA by mutating the gene from which the mRNA is transcribed. Mutating the gene may comprise deleting the sequence of the tRNA-like structure from the gene, mutating the sequence of the tRNA-like structure to change the tridimensional configuration thereof, or inserting a genetic element (such as, for example, a transposon, a retrotransposon, a T-DNA insertion, or a retroviral repeat sequence) into the gene to remove the tRNA-like structure from the transcribed part of the gene, such that the sequence encoding the TLS is no longer transcribed as part of the mRNA to which it initially belonged, thus changing the mobility of that mRNA.
Deleting the sequence of the tRNA-like structure from the gene may, for example, be accomplished by using genome editing tools such as CRISPR-Cas, using two guide RNAs designed to respectively target sequences upstream and downstream of the coding sequence of the tRNA-like structure in the gene, thereby leading to double-stranded breaks in the gene up- and downstream from the coding sequence of the tRNA-like structure in the gene, which may result in the removal of said coding sequence of the tRNA-like structure from the gene. Alternatively, deleting the sequence of the tRNA-like structure from the gene may be accomplished in vitro, using standard molecular biology techniques or gene synthesis techniques, and subsequently introducing a DNA construct encoding a tRNA::mRNA bicistronic transcript, or the in vitro transcribed transcript itself, into the organism.
The tridimensional configuration of a tRNA typically comprises stem-loop structures: from 5′ to 3′ these are the D arm (or D stem-loop), the A or anticodon arm (or A stem-loop, or anticodon stem-loop), and the T arm (or TψC arm, TψC arm, or TψC stem-loop). Between the A and T arms a variable loop may be present. Mutating the sequence of the tRNA-like structure to change the tridimensional configuration thereof may comprise deleting part of the transcribed sequence thereof, mutating one or more nucleotides involved in the formation of a stem-loop structure in the tRNA-like structure, or inserting one or more nucleotides in a stem-loop of the tRNA-like structure.
Mutations may be induced at random or in a targeted manner, using various mutagenesis methods known in the prior art. In vitro mutagenesis methods include, for example, site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis. In vivo mutagenesis may, for example, be achieved randomly by means of one or more chemical compounds, such as ethyl methanesulphonate, nitrosomethylurea, hydroxylamine, proflavine, N-methyl-N-nitrosoguanidine, N-ethyl-N-nitrosourea, N-methyl-N-nitro-nitrosoguanidine, diethyl sulphate, ethylene imine, sodium azide, formaline, urethane, phenol, ethylene oxide, and/or by physical means, such as UV-irradiation, fast-neutron exposure, X-rays, gamma irradiation, and/or by insertion of genetic elements, such as transposons, T-DNA, retroviral elements. Mutations may also be introduced specifically by means of homologous recombination, oligonucleotide-based mutation induction, or targeted genome editing methods such as TALEN, zinc-finger technology, or CRISPR-Cas.
In one embodiment, deleting part of the transcribed sequence of the tRNA-like structure in a gene comprises deleting the A and/or T stem-loops from the tRNA-like structure. As is shown in Example 2, removal of the A and T stem-loop structures results in the abolition of mobility of an mRNA transcript that is mobile when the A and T stem-loop structures are present. The removal of the D stem-loop, of the D and T stem-loops or of the D and A stem-loops from a gene did not abolish the mobility of the same mRNA transcript.
It should be noted that the term “tRNA-like structure” as used in this application not only comprises intact tRNA structures that are present in the mRNA transcript of a gene (and which can be screened for using the tRNA motif scan approach, explained in Example 2), but also incomplete tRNA structures that lack one or more stem-loop structures but that still retain the ability to enable the intercellular mobility of the mRNA transcript of said gene. The term “tRNA-like structure” as used in the context of the present invention thus also encompasses tRNA structures that lack the D stem-loop, the D and T stem-loops, or the D and A stem-loops. The efficacy of such incomplete structures for conferring mobility to an mRNA transcript has been shown in Example 2 and FIG. 4C.
In one embodiment, the present invention relates to a method for changing the intercellular mobility of an mRNA of a gene in an organism, wherein the change in mobility results in a loss of the function of the gene. This is the case for genes whose mRNA transcript is transcribed in one cell-type, tissue or organ of an organism, but whose gene product (the encoded protein) functions in at least one other cell-type, tissue or organ of the organism, where its mRNA transcript is not produced. For such genes, the intercellular mobility of their mRNA transcript is thus essential for the normal functionality of the gene in the organism. The loss of intercellular mobility of the mRNA transcript of such a gene results in a loss of the function of the gene in the cells, tissues or organs towards which the mRNA transcript moves during normal growth, development and/or functioning of the organism and in which it is normally translated into protein. The loss of intercellular mobility of the mRNA transcript thus leads to the absence or reduced presence of the gene product (i.e. the encoded protein) in the cells, tissues or organs where the mRNA transcript moves towards during normal growth, development and/or functioning of the organism. The phenotype of said cells, tissues or organs may be affected by this absence or reduced presence, and the morphology, responsiveness and/or functionality of said cells, tissues or organs may be affected thereby. This situation is illustrated in Example 3, which shows that the loss of transcript mobility for the CK1 gene in plants results in the absence of CK1 transcript in the phloem, and in a very similar phenotype as could be observed in a knock-out mutant that lacked the CK1 transcript altogether.
In another embodiment, the present invention relates to a method for changing the intercellular mobility of an mRNA of a gene in an organism, comprising including the coding sequence of a tRNA-like structure in the transcribed part of the gene.
In one embodiment, the present invention relates to a method for changing the intercellular mobility of an mRNA of a gene in an organism, wherein the change in mobility results in ectopic presence of the gene product in the organism. In this manner, an mRNA transcript that does not display intercellular mobility can be rendered mobile, by including a tRNA-like structure in said transcript.
In another embodiment, the present invention relates to a method for changing the intercellular mobility of an mRNA of a gene in an organism, wherein the gene is a non-endogenous gene in at least part of the organism. This situation is illustrated in Example 2, wherein the addition of a TLS to the 3′UTR of the β-glucuronidase-encoding gene (GUS) makes the GUS transcript mobile, and leads to translation of the protein in distant parts of the plant, across a graft junction. This situation is also illustrated in Example 1, wherein a modified version of DMC1 is provided in the form of a DNA construct in a transgenic rootstock. The modification comprise the addition of a TLS in the 3′UTR of the gene, and a truncation of the gene which turns the encoded protein into a dominant-negative version that interferes with the function of wild-type DMC1. Said modified transcript is demonstrated to be transported towards the male spores in a grafted scion, where the encoded protein (which is a dominant-negative version of DMC1) subsequently affects development.
Suitably, including the coding sequence of a tRNA-like structure in the transcribed part of the gene is achieved by introducing the complementary sequence of the tRNA-like structure in a DNA construct comprising the complementary sequence of the mRNA, or by introducing the complementary sequence of the tRNA-like structure in the endogenous gene, by means of e.g. genome editing tools such as CRISPR-Cas.
In one embodiment, the coding sequence of a tRNA-like structure is introduced into the 3′ untranslated region (3′UTR) of the mRNA of the gene. Preferably, the coding sequence of a tRNA-like structure is then introduced after the stop-codon of a gene, such that it is present in the transcribed part of the gene but does not affect the open reading frame of the gene. Alternatively, the coding sequence of a tRNA-like structure may also be introduced into the 5′ untranslated region (5′UTR) of the mRNA of the gene, or in the open reading frame of the gene, in such a way that the coding sequence is not disrupted or the reading frame is shifted.
The DNA construct may be stably integrated in the genome of the organism, transiently expressed in the organism, or in vitro transcribed. Said DNA construct comprises a suitable promoter that is capable of driving expression of the gene in a suitable environment. There are, for example, promoters that are ubiquitous, organ-, tissue- or cell-type specific, and promoters that are constitutive or regulated by environmental (physical or biological) or endogenous (developmental) queues, or regulated by chemicals.
Suitable constitutive promoter control sequences for mammals include, but are not limited to, cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor (ED1)-alpha promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, fragments thereof, or combinations of any of the foregoing.
Suitable constitutive promoter control sequences for plants include, but are not limited to, 35S cauliflower mosaic virus (CaMV) promoter, opine promoters, ubiquitin promoters, actin promoters, tubulin promoters, alcohol dehydrogenase promoters, fragments thereof, or combinations of any of the foregoing.
Examples of suitable regulated promoter control sequences for animals or plants include, without limit, those regulated by heat shock, cold, drought, heavy metals, steroids (such as dexamethasone, β-estradiol), antibiotics, or alcohols (such as ethanol).
Non-limiting examples of tissue-specific mammalian promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GP11b promoter, ICAM-2 promoter, INF-13 promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.
Non-limiting examples of tissue-specific plant promoters include the β-phaseolin, glutenin, a-Kaf, zein, β-conglycinin and AGPase promoters (for seed-specific expression), the LAT52, GEX2, MANS and FRK4 promoters (for pollen-specific expression), the rolD, REO, Prx and Tip2 promoters (for root-specific expression), and the Zmglp1, PnGLP and PDX1 promoters (for leaf-specific expression).
The promoter sequence can be wild type or it can be modified for more efficient or efficacious expression. In one exemplary embodiment, the encoding DNA can be operably linked to a CMV promoter for constitutive expression in mammalian cells.
In certain embodiments, the sequence of an endogenous or non-endogenous gene can be operably linked to a promoter sequence that is recognised by a phage RNA polymerase for in vitro mRNA synthesis. In such embodiments, the in vitro-transcribed RNA can be purified for administration to an organism. For example, the promoter sequence can be a T7, T3, or SP6 promoter sequence or a variation of a T7, T3, or SP6 promoter sequence. In an exemplary embodiment, the DNA encoding the fusion protein is operably linked to a T7 promoter for in vitro mRNA synthesis using T7 RNA polymerase.
In alternative embodiments, the sequence of an endogenous or non-endogenous gene can be operably linked to a promoter sequence for in vitro expression in bacterial or eukaryotic cells.
The DNA construct or the in vitro synthesised RNA transcript may be introduced into the organism by various methods known in the prior art.
When a DNA construct is intended for stable integration in the genome of an organism or for transient expression in an organism, it can be introduced into said organism or into a cell derived therefrom by means of (micro)injection, ingestion, transfection, electroporation, DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection, protein transduction domain mediated transduction, virus-mediated gene delivery, PEG-mediated transfection of protoplasts, infection with a suitable virus or bacterium (such as Agrobacterium for plants), etcetera.
When a DNA construct is intended for in vitro transcription of the encoded mRNA, in vitro transcription may be achieved using any in vitro transcription system known in the art. Alternatively, transcription may also be done in a heterologous organism, after which the transcript of interest is subsequently purified for further use.
An in vitro synthesised RNA transcript may be introduced into the organism or into a cell derived therefrom by the methods listed above (such as injection into the blood stream of a mammal or into the phloem of a plant), and also by means of, for example, exogenous application on the surface of the organism. Exogenous application may for example be performed according to the disclosure in patent application WO2015/200539. Exogenous application to cell cultures or tissue cultures may, for example, be performed using electroporation, transfection, etcetera.
When an in vitro synthesised RNA transcript is introduced into an organism, a suitable promoter is not required, because transcription does not need to take place inside the organism's cells. Said transcript may immediately be translated, and/or it may move into cells of the organism and from one cell of the organism to other cells of the organism. According to the invention the movement of the transcript is due to the presence of TLS.
Another application is the use of cultured cells that produce an mRNA transcript harbouring a TLS for in vivo application in an organism. For example, cultured cells harbouring a gene that—when transcribed—gives rise to an mRNA transcript harbouring a TLS, may be injected (or otherwise administered) into a mammal's blood stream, where said cells secrete an mRNA transcript harbouring a TLS into the extracellular matrix. This mRNA transcript can subsequently be taken up by other cells in the organism, due to the presence of a TLS. This constitutes an efficient way of delivering mRNA transcripts in vivo. Said mRNA transcript may encode any protein that can be administered to the organism. In a further embodiment, said mRNA transcript encodes a therapeutic protein, an endogenously occurring protein or a modified version thereof, or a non-endogenously occurring protein. Suitably, the administration of said cells to the organism is for the treatment of a disease, or for gene therapy.
In one embodiment, the complementary sequence of a tRNA-like structure is introduced into the transcribed part of a gene in a DNA construct, and the gene comprising the tRNA-like structure is transcribed in vitro. The mRNA transcript comprising the tRNA-like structure is subsequently introduced into an organism, cell culture or tissue culture, such that it can move into cells, and from one cell into other cells. This embodiment represents an application of the present invention that does not require the use of DNA constructs in vivo. The manner in which the mRNA transcript comprising the tRNA-like structure is introduced into an organism, cell culture or tissue culture, depends on the type of organism, cell culture or tissue culture that is being used. Means by which introduction can be achieved comprise, for example, injection into the blood stream of animals, injection into the vascular tissue or apoplast of plants, or exogenous application on the surface of the organism. The use of cell-penetrating peptides may for example facilitate the entry of the mRNA transcript into the organism.
As stated above, the term “tRNA-like structure” as used in this application not only comprises intact tRNA structures that are present in the mRNA transcript of a gene (and which can be screened for using the tRNA motif scan approach, explained in Example 2), but also incomplete tRNA structures that lack one or more stem-loop structures but that still retain the ability to enable the intercellular mobility of the mRNA transcript of said gene. The term “tRNA-like structure” as used in the context of the present invention thus also encompasses tRNA structures that lack the D stem-loop, the D and T stem-loops, or the D and A stem-loops. The term “tRNA-like structure” as used in this application thus encompasses intact tRNA structures and tRNA structures that comprise at least the A stem-loop and/or the T stem-loop.
The efficacy of such incomplete structures for conferring mobility to an mRNA transcript has been shown in Example 2 and FIG. 4C. In one embodiment, the tRNA-like structure is selected from the group consisting of tRNAAla, tRNAArg, tRNAAsn, tRNAAsp, tRNACys, tRNAGln, tRNAGlu, tRNAGly, tRNAHis, tRNAIle, tRNALeu, tRNALys, tRNAMet, tRNAPhe, tRNAPro, tRNASer, tRNAThr, tRNATrp, tRNATyr, tRNAval. In another embodiment, the tRNA-like structure is selected from the group consisting of tRNAAla, tRNAArg, tRNAAsn, tRNAAsp, tRNACys, tRNAGln, tRNAGlu, tRNAGly, tRNAHis, tRNAIle, tRNALeu, tRNALys, tRNAMet, tRNAPhe, tRNAPro, tRNASer, tRNAThr, tRNATrp, tRNATyr, tRNAVal and it lacks the D stem-loop, the D and T stem-loops, or the D and A stem-loops.
In a further embodiment, the tRNA-like structure comprises a tRNA anticodon with a sequence from 5′ to 3′ in the transcribed mRNA selected from the group consisting of AGC, CGC, UGC, ACG, CCG, CCU, UCG, UCU, GUU, GUC, GCA, CUG, UUG, CUC, UUC, ACC, CCC, GCC, UCC, GUG, AAU, AAG, CAA, CAG, GAG, UAA, UAG, CUU, UUU, CAU, GAA, AGG, CGG, UGG, AGA, CGA, GCU, GGA, UGA, AGU, CGU, UGU, CCA, GUA, AAC, CAC, UAC. In another embodiment, the tRNA-like structure comprises a tRNA anticodon with a sequence from 5′ to 3′ in the transcribed mRNA selected from the group consisting of AGC, CGC, UGC, ACG, CCG, CCU, UCG, UCU, GUU, GUC, GCA, CUG, UUG, CUC, UUC, ACC, CCC, GCC, UCC, GUG, AAU, AAG, CAA, CAG, GAG, UAA, UAG, CUU, UUU, CAU, GAA, AGG, CGG, UGG, AGA, CGA, GCU, GGA, UGA, AGU, CGU, UGU, CCA, GUA, AAC, CAC, UAC and it lacks the D stem-loop, or the D and T stem-loops.
As shown in Example 2, some tRNA-like structures exist that have a tridimensional structure that does not promote intercellular mobility of an mRNA transcript, such as tRNAIle with anticodon UAU.
In one embodiment, the present invention relates to a method for changing the intercellular mobility of an mRNA of a gene in a plant, wherein said plant consists of a rootstock of a first plant upon which a scion of a second plant has been grafted, and wherein the intercellular mobility of an mRNA of a gene in the first and/or the second plant has been changed. This embodiment has been illustrated in Examples 1-3, with Arabidopsis and Nicotiana (tobacco).
This embodiment can be carried out with any plant species for which grafting is possible. Commercially relevant plants that can be grafted are, for example, plants belonging to the genera Apium, Beta, Brassica, Capsicum, Cichorium, Citrullus, Cucumis, Cucurbita, Benincasa, Daucus, Eruca, Lactuca, Lagenaria, Luffa, Phaseolus, Pisum, Lens, Raphanus, Solanum, Spinacia, Valerianella, Nicotiana, Petunia, Arabidopsis, Capsella, Arabis, Cardamine, Malus, Pyrus, Prunus, Vitis, Rosa, Fragaria, Populus, Fagus, Pinus, Picea, Ginkgo, Larix, Betula, Quercus, Salix, Alnus, Corylus, Amygdalus, Vaccinium, Rubus, Persea, Citrus, Castanea, Acer, Fraxinus, Coffea, Camellia, Theobroma, Olea, Cicer, Juglans, Pistacia, Arachis, Anacardium, Macadamia, Ficus, Litchi, Actinidia, Bougainvillea, Helianthus, Hibiscus, Malva, Gossypium, Cannabis, Stevia, Opuntia, and Ipomoea. This is of course a non-exhaustive list.
In general, many dicotyledonous plants can be grafted, whereas monocotyledonous plants cannot be grafted. However, a method has recently been developed to graft recalcitrant species such as soybean (Glycine max) and commercially important species of the Poaceae family, such as maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), etcetera (T. Harada, European patent application EP2918161). For those species this embodiment of the method of the present invention can thus also be applied.
This embodiment has various distinct applications. Firstly, the intercellular mobility of an mRNA of an endogenous gene may be changed. This may be done by changing the encoding gene in the rootstock or in the scion of a grafted plant, and it may either lead to a loss of mobility for an mRNA transcript that is naturally mobile in the plant, or to a gain of mobility for an mRNA transcript that is naturally non-mobile in the plant. In case of a gain of mobility, the mRNA transcript may move across the graft junction. In case of a loss of mobility, the rootstock or scion may be depleted of the mRNA transcript, which may lead to phenotypic effects. Changing the encoding gene can be done in all manners that have been described above, such as mutagenesis, genome editing and/or the use of transgenes.
In a further embodiment, the phenotype of the rootstock and/or scion is changed. This may for example be achieved by the movement of a mobile mRNA transcript across the graft junction, where it complements for the absence of that mRNA transcript in either the rootstock or the scion due to a mutation. This thus represents a method for mutant complementation, by grafting a mutant plant onto a wild-type plant and rescuing the mutant phenotype by means of a mobile mRNA transcript that compensates for the absence of the corresponding endogenous mRNA transcript.
In addition to changing the intercellular mobility of an mRNA of an endogenous gene, it is of course possible to induce additional mutations in said mRNA transcript, such as point mutations, or to induce other modifications in said mRNA transcripts, such as insertions or deletions. Such additional mutations or modifications may, for example, have an effect on the primary sequence of the encoded protein, and on the encoded protein's structure and/or function. An example of this embodiment is presented in Example 1, wherein a modified (dominant-negative) form of DMC1 is used to interfere with DMC1 function in male spores.
Secondly, in another embodiment, a non-endogenous gene may be rendered mobile in an organism. The use of non-endogenous genes, such as reporter genes, is common practice in molecular biology, but the expression pattern of such non-endogenous genes depends entirely on the use of a suitable promoter, that is able to drive a gene's expression in the organism. Suitable promoters have been discussed above in this text. This situation is illustrated in Example 2, with β-glucuronidase (GUS).
Another example of a non-endogenous gene for which it is interesting to render its mRNA transcript mobile in an organism is Cas9. Cas9 is an RNA-guided endonuclease that has the capacity to create double-stranded breaks in DNA in vitro and in vivo, also in eukaryotic cells. It is part of an RNA-mediated adaptive defence system known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in bacteria and archaea. Cas9 gets sequence-specificity when it associates with a guide RNA molecule, which can target sequences present in an organism's DNA based on their sequence. Cas9 requires the presence of a Protospacer Adjacent Motif (PAM) immediately following the DNA sequence that is targeted by the guide RNA. The Cas9 enzyme has been first isolated from Streptococcus pyogenes (SpCas9), but functional homologues from many other bacterial species have been reported, such as Neisseria meningitides, Treponema denticola, Streptococcus thermophilus, Francisella novicida, Staphylococcus aureus, etcetera. For SpCas9, the PAM sequence is 5′-NGG-3′, whereas various Cas9 proteins from other bacteria have been shown to recognise different PAM sequences. In nature, the guide RNA is a duplex between crRNA and tracrRNA, but a single guide RNA (sgRNA) molecule comprising both crRNA and tracrRNA has been shown to work equally well (Jinek et al, 2012, Science 337: 816-821). The advantage of using an sgRNA is that it reduces the complexity of the CRISPR-Cas9 system down to two components, instead of three. For use in an experimental setup (in vitro or in vivo) this is an important simplification.
An alternative for Cas9 is, for example, Cpf1, which does not need a tracrRNA to function, which recognises a different PAM sequence, and which creates sticky end cuts in the DNA, whereas Cas9 creates blunt ends.
On the one hand, genetic modification techniques can be applied to express an RNA-guided endonuclease and/or guide RNAs in eukaryotic cells. One or more DNA constructs encoding an RNA-guided endonuclease and at least one guide RNA can be introduced into a cell or organism by means of stable transformation (wherein the DNA construct is integrated into the genome) or by means of transient expression (wherein the DNA construct is not integrated into the genome, but it expresses an RNA-guided endonuclease and at least one guide RNA in a transient manner). This approach requires the use of a transformation vector and a suitable promoter for expression in said cell or organism. Organisms into which foreign DNA has been introduced are considered to be Genetically Modified Organisms (GMOs), and the same applies to cells derived therefrom and to offspring of these organisms. In important parts of the worldwide food market, transgenic food is not allowed for human consumption, and not appreciated by the public. There is therefore a need for an alternative, “DNA-free” delivery method of CRISPR-Cas components into intact plants, that does not involve the introduction of DNA constructs into the cell or organism.
In the prior art, introducing the mRNA encoding Cas9 into a cell or organism has been described, after in vitro transcription from a DNA construct encoding an RNA-guided endonuclease, together with at least one guide RNA. This approach does not require the use of a transformation vector and a suitable promoter for expression in said cell or organism.
Another known approach is the in vitro assembly of ribonucleoprotein (RNP) complexes, comprising an RNA-guided endonuclease protein (for example Cas9) and at least one guide RNA, and subsequently introducing the RNP complex into a cell or organism. The GMO status of this approach is not yet clear, but the introduction of RNP complexes into intact organisms is technically challenging. In animals and animal cell and tissue cultures, RNP complexes have been introduced by means of, for example, injection, electroporation, nanoparticles, vesicles, and with the help of cell-penetrating peptides. However, the RNP complexes can only perform their function in cells where they have been introduced into, and this limits the efficiency of this approach. There is a need for a self-propagating system wherein the CRISPR-Cas components move between cells.
In plants, the use of RNPs has been demonstrated in protoplasts, for example with polyethylene glycol (PEG) transfection (Woo et al., 2015, Nat. Biotech. 33: 1162-1164). However, the applicability of this technique depends entirely on the availability of protocols for the regeneration of entire plants from protoplasts. Such protocols are not available for all plants, and there is therefore a need for an alternative delivery method of CRISPR-Cas components in plant cells and in intact plants.
A specific application of the present invention involves the mobilisation of an RNA-guided endonuclease-encoding mRNA in a plant. A shown in Example 1 with DMC1-encoding mRNA, mRNA transcripts that are present in the phloem of a plant are able to reach the plant's male spores. When a similar approach is taken with an RNA-guided endonuclease-encoding mRNA, it is therefore not only possible to modify the genome of a plant's somatic cells, but also the genome of its male germline, in the presence of at least one suitable guide RNA. The latter category of genome modifications is heritable, as they can be transmitted to the next generation.
Suitably, in one embodiment, the RNA-guided endonuclease gene can be encoded by a DNA construct that is integrated into the genome of one plant, onto which another plant is subsequently grafted. In this manner, the cells of the rootstock harbour a transgenic construct, but the cells of the scion do not. However, when the mRNA transcript encoding the RNA-guided endonuclease comprises a TLS, it is rendered mobile and it is able to cross the graft junction, and the RNA-guided endonuclease-encoding transcript can be delivered into cells of the scion, where the RNA-guided endonuclease protein can be translated. In this situation, cells of the scion produce a protein that is not encoded in their own genome, and this protein is capable of modifying a chromosomal sequence in said cells, when at least one suitable guide RNA is present in said cells.
Alternatively, the mRNA transcript encoding the RNA-guided endonuclease and comprising a TLS may be introduced into a plant by direct injection, for example into the vascular tissue, or by exogenous application, as has been described above. Said transcript will then behave as if it had been produced inside the plant, and it will be delivered into cells of the plant, where the encoded protein will be translated and where it will perform its function. In this embodiment, grafting is not a requirement.
After said modification of a chromosomal sequence has taken place, the cells can be used to produce plants that harbour said modification in their genome, using any plant regeneration method known in the art (such as in vitro tissue culture).
In a preferred embodiment, the RNA-guided endonuclease-encoding mRNA transcript is translated into the encoded protein inside male spores of a plant, where the encoded protein may modify a chromosomal sequence in the presence of at least one suitable guide RNA. The at least one guide RNA may, for example, be introduced into the cells by means of injection into the anther, the phloem or other parts of the plant, Agrobacterium-mediated transformation, or exogenous application, or it may be encoded by a DNA-construct that is present in the cells of the plant that produces said male spores. Said spores may be produced by the scion of a grafted plant. It has been shown that many small RNA molecules (of comparable size as a typical guide RNA) are mobile in the phloem and are able to move systemically throughout the plant and across graft junctions. It has also been shown that such small RNA molecules are able to reach the male spores of a scion when they are transcribed in a rootstock (see for example patent application WO2013017683).
In a particular embodiment the invention thus provides a method for modifying a chromosomal sequence in a plant cell or spore, comprising:
a) combining a first plant comprising a rootstock, which harbours a nucleic acid sequence encoding a modified, non-naturally occurring RNA-guided endonuclease protein, with a scion from a second plant grafted onto the rootstock of the said first plant, whereby the said nucleic acid sequence generates a transcript in the rootstock of the first plant, which transcript is transported systemically across the graft junction to enter the scion of the second plant, and which transcript is imported into cells of the scion of the second plant, where it is translated into a functional protein;
b) providing at least one guide RNA or DNA encoding at least one guide RNA to at least one cell or spore of the scion of the second plant, wherein the at least one guide RNA is a single molecule comprising a 5′ region that is complementary to a target site in a chromosomal sequence of said second plant;
c) allowing each guide RNA to direct an RNA-guided endonuclease protein to a targeted site in the chromosomal sequence, where the RNA-guided endonuclease protein introduces a double-stranded break in the targeted site, and allowing the cell to repair the double-stranded break by a DNA-repair process such that the chromosomal sequence is modified in at least one cell or spore of the scion of the second plant.
The nucleic acid sequence encoding a modified, non-naturally occurring RNA-guided endonuclease protein may be a transgene that is stably integrated or transiently expressed.
In a particular embodiment, the RNA-guided endonuclease protein is derived from a Cas protein. The Cas protein is suitably Cas9, or a variant thereof. The Cas protein is for example derived from the genus Streptococcus. 
In a particular embodiment, the nucleic acid sequence encoding a modified, non-naturally occurring RNA-guided endonuclease protein comprises a tRNA-like structure that allows the transcript to be transported systemically across the graft junction from the rootstock of the first plant into the scion of the second plant. The nucleic acid sequence encoding a modified, non-naturally occurring RNA-guided endonuclease protein may further comprise at least one localisation signal to a DNA-containing organelle and optionally a marker domain. Non-limiting examples of marker domains are fluorescent markers (such as Green Fluorescent Protein, Yellow Fluorescent Protein, mCherry), epitope tags (such as FLAG, His, HA, calmodulin), etcetera.
The localisation signal is suitably selected from the group comprising a nuclear localisation signal, a chloroplast targeting signal, a mitochondrial targeting signal.
The nucleic acid sequence encoding a modified, non-naturally occurring RNA-guided endonuclease protein may optionally further comprises a Fold endonuclease domain or another protein domain.
The tRNA-like structure can be tRNAAla, tRNAArg, tRNAAsn, tRNAAsp, tRNACys, tRNAGln, tRNAGlu, tRNAGly, tRNAHis, tRNAIle, tRNALeu, tRNALys, tRNAMet, tRNAPhe, tRNAPro, tRNASer, tRNAThr, tRNATrp, tRNATyr, tRNAVal. The term “tRNA-like structure” as used in the context of the present invention also encompasses tRNA structures that lack the D stem-loop, the D and T stem-loops, or the D and A stem-loops.
The tRNA-like structure is located in the 3′UTR of the transcript, in the 5′UTR of the transcript, or in the coding sequence of the transcript.
In one embodiment, the guide RNA is a single-chain guide RNA, wherein the guide RNA is a DNA-targeting RNA comprising a) a first segment comprising a nucleotide sequence that is complementary to a chromosomal sequence in a plant cell or spore, and b) a second segment that interacts with an RNA-guided endonuclease protein. Suitably, the guide RNA is a single-chain guide RNA comprising a crRNA and a tracrRNA.
Expression of a transgenic nucleic acid sequence in the first plant is suitably achieved by operably linking the said nucleic acid sequence to a promoter sequence that confers a ubiquitous expression profile or a tissue- or cell-type specific expression profile onto the transgenic nucleic acid sequence, and/or to an inducible promoter. Suitable promoters have been discussed above.
In one embodiment, the promoter sequence confers onto the transgenic nucleic acid sequence an expression profile that encompasses roots.
When expression of the transgenic nucleic acid sequence in the first plant is transient, transient expression is suitably achieved by use of an inducible promoter, selected from the group comprising heat-inducible promoters, cold-inducible promoters, chemical-inducible promoters, steroid-inducible promoters and alcohol-inducible promoters. Suitable promoters have been discussed above.
Providing the at least one guide RNA or DNA encoding the at least one guide RNA to at least one cell or spore of the scion of the second plant is suitably accomplished by means of injection, Agrobacterium-mediated transformation, exogenous application, or stable integration or transient expression of a DNA-construct.
The invention further relates to such a method, wherein exogenous application of the at least one guide RNA or DNA encoding the at least one guide RNA comprises applying onto the scion of the second plant or a part thereof a mixture comprising:
a) a cationic polyelectrolyte;
b) an osmolyte; and
c) the at least one guide RNA or DNA encoding the at least one guide RNA, wherein the at least one guide RNA is a single molecule comprising a 5′ region that is complementary to a target site in a chromosomal sequence of said second plant. Exogenous application may be performed according to the disclosure in patent application WO2015/200539.
In one embodiment of this latter method a chromosomal sequence of at least one cell or spore of the second plant is genetically modified without the insertion of external genetic material.
In a particular embodiment of this latter method, the first plant and the second plant belong to the same plant family. Suitably, the first plant and the second plant belong to the same genus, in particular to the same plant species.
In a further embodiment, the plant belongs to one of the following genera: Beta, Brassica, Capsicum, Cichorium, Citrullus, Cucumis, Cucurbita, Benincasa, Daucus, Eruca, Lactuca, Lagenaria, Luffa, Phaseolus, Pisum, Raphanus, Solanum, Spinacia, Valerianella, Nicotiana, Petunia, Arabidopsis, Capsella, Arabis, Malus, Pyrus, Prunus, Vitis, Rosa, Fragaria, Populus, Fagus, Pinus, Picea, Ginkgo, Larix, Betula, Quercus, Salix, Alnus, Corylus, Amygdalus, Vaccinium, Rubus, Persea, Citrus, Castanea, Acer, Fraxinus, Coffea, Camellia, Theobroma, Olea, Cicer, Juglans, Pistacia, Arachis, Anacardium, Macadamia, Ficus, Litchi, Actinidia, Bougainvillea, Helianthus, Hibiscus, Malva, Glycine, Gossypium, Cannabis, Stevia, Opuntia, or Ipomoea. 
In an embodiment of this invention where grafting is not performed (for example when the guide RNA and the RNA-guided endonuclease-encoding transcript are both administered to the plant by means of a DNA-free delivery method, such as, for example, injection of the guide RNA and the RNA-guided endonuclease-encoding mRNA transcript into the phloem of the plant), the plant may belong to one of the following genera: Allium, Apium, Beta, Brassica, Capsicum, Cichorium, Citrullus, Cucumis, Cucurbita, Benincasa, Daucus, Eruca, Lactuca, Lagenaria, Luffa, Phaseolus, Pisum, Lens, Raphanus, Solanum, Spinacia, Valerianella, Nicotiana, Petunia, Arabidopsis, Capsella, Arabis, Cardamine, Malus, Pyrus, Prunus, Vitis, Rosa, Fragaria, Populus, Fagus, Pinus, Picea, Ginkgo, Larix, Betula, Quercus, Salix, Alnus, Corylus, Amygdalus, Vaccinium, Rubus, Persea, Citrus, Castanea, Acer, Fraxinus, Coffea, Camellia, Theobroma, Olea, Cicer, Juglans, Pistacia, Arachis, Anacardium, Macadamia, Ficus, Litchi, Actinidia, Bougainvillea, Helianthus, Hibiscus, Malva, Glycine, Gossypium, Cannabis, Stevia, Opuntia, Ipomoea, Manihot, Humulus, Acacia, Medicago, Trifolium, Lotus, Vicia, Linum, Fagopyrum, Zea, Triticum, Avena, Hordeum, Oryza, Zizania, Secale, Triticosecale, Sorghum, Bambusa, Dendrocalamus, Saccharum, Cymbopogon, Pennisetum, Panicum, Festuca, Lolium, Phleum, Poa, Miscanthus, Asparagus, Agave, Yucca, Cocos, Elaeis, Phoenix, Amaryllis, Narcissus, Aloe, Canna, Iris, Colchicum, Crocus, Gladiolus, Juncus, Lilium, Tulipa, Musa, Dendrobium, Phalaenopsis, Vanilla, Typha, Zingiber, Curcuma, Lemna. 